Stimulation of angiogenesis via enhanced endothelial expression of syndecan-4 core

ABSTRACT

The present invention provides tangible means and methods for stimulation of angiogenesis via enhanced endothelial expression of core proteins having a syndecan-4 cytoplasmic region intracellularly. The tangible means include a prepared DNA sequence fragment having separate and individual DNA sequenced portions coding for an heparan sulfate binding extracellular domain, a central transmembrane domain, and a cytoplasmic domain coding for the syndecan-4 polypeptide. The prepared DNA sequence unitary fragment may be delivered to endothelial cells in-situ, both under in-vivo and/or in-vitro conditions, using suitable expression vectors including plasmids and viruses. The resulting transfected endothelial cells overexpress heparan sulfate binding, core proteins; and the resulting overexpression of these proteoglycan entities causes stimulation of angiogenesis in-situ.

FIELD OF THE INVENTION

The present invention is concerned generally with the stimulation ofangiogenesis in-situ in living tissues and organs; and is particularlydirected to the preparation and use of prepared DNA sequences andexpression vectors suitable for transfection of endothelial cellsin-situ such that overexpression of extracellular matrix heparin sulfatebinding proteoglycans subsequently occurs in-situ.

BACKGROUND OF THE INVENTION

Angiogenesis, by definition, is the formation of new capillaries andblood vessels within living tissues; and is a complex process firstrecognized in studies of wound healing and then within investigations ofexperimental tumors. Angiogenesis is thus a dynamic process whichinvolves extracellular matrix remodeling, endothelial cell migration andproliferation, and functional maturation of endothelial cells intomature blood vessels [Brier, G. and K. Alitalo, Trends Cell Biology 6:454-456 (1996)]. Clearly, in normal living subjects, the process ofangiogenesis is a normal host response to injury, and as such, is anintegral part of the host body's homeostatic mechanisms.

It will be noted and appreciated, however, that whereas angiogenesisrepresents an important component part of tissue response to ischemia,or tissue wounding, or tumor-initiated neovascularization, relativelylittle new blood vessel formation or growth takes place in most livingtissues and organs in mature adults (such as the myocardium of theliving heart) [Folkman, J. and Y. Shing, J. Biol. Chem. 267: 10931-10934(1992); Folkman, J., Nat Med. 1: 27-31 (1995); Ware, J. A. and M.Simons, Nature Med. 3: 158-164 (1997)]. Moreover, although regulation ofan angiogenetic response in-vivo is a critical part of normal andpathological homeostasis, little is presently known about the controlmechanisms for this process. A number of different growth factors andgrowth factor receptors have been found to be involved in the process ofstimulation and maintenance of angiogenetic responses. In addition, anumber of extracellular matrix components and cell membrane-associatedproteins are thought to be involved in the control mechanisms ofangiogenesis. Such proteins include SPARC [Sage etal., J. Cell Biol.109: 341-356 (1989); Motamed, K. and E. H. Sage, Kidney Int. 51:1383-1387 (1997)]; thrombospondin 1 and 2 respectively [Folkman, J.,Nat. Med. 1: 27-31 (1995); Kyriakides et al., J. Cell Biol. 140: 419-430(1998)]; and integrins αvβ5 and αvβ3[Brooks et al., Science 24: 569-571(1994); Friedlander et al., Science 270: 1500-1502 (1995)]. However, itis now recognized that a major role is played by heparan-binding growthfactors such as basic fibrocyte growth factor (bFGF) and vascularendothelial growth factor (VEGF); and thus the means for potentialregulation of angiogenesis involves the extracellular heparan sulfatematrix on the surface of endothelial cells.

Research investigations have shown that heparan sulfate core proteins orproteoglycans mediate both heparin-binding growth factor/receptorinteraction at the cell surface; and that accumulation and storage ofsuch growth factors within the extracellular matrix proper occurs[Vlodavsky et al., Clin. Exp. Metastasis 10: 65 (1992); Olwin, B. B. andA. Rapraeger, J. Cell Biol. 118: 631-639 (1992); Rapraeger, A. C., Curr.Opin. Cell Biol. 1: 844-853 (1993)]. The presence of heparin or heparansulfate is required for bFGF-dependent activation of cell growthin-vitro [Yayon et al., Cell 64: 841-848 (1991); Rapraeger et al.,Science 252: 1705-1708 (1991)]; and the removal of heparan sulfatechains from the cell surface and extracellular matrix by enzymaticdigestion greatly impairs bFGF activity and inhibits neovascularizationin-vivo [Sasisekharan et al., Proc. Natl. Acad. Sci. USA 21: 1524-1528(1994)]. Ample scientific evidence now exists which demonstrates thatany alteration of heparan sulfate (HS) chain composition on the cellsurface or within the extracellular matrix which is initiated by meansof an altered synthesis, or a degradation, or a substantive modificationof glycosaminoglycan (GAG) chains can meaningful affect theintracellular signaling cascade initiated by the growth factor. Theimportance of heparan sulfate in growth factor-dependent signaling hasbecome well recognized and established in this field.

Heparan sulfate (HS) chains on the cell surface and within theextracellular matrix are present via binding to a specific category ofproteins commonly referred to as “proteoglycans”. This category isconstituted of several classes of core proteins, each of which serve asacceptors for a different type of glycosaminoglycan (GAG) chains. TheGAGs are linear co-polymers of N-acetyl-D-glycosamine [binding heparansulfate] or N-acetyl-D-galactosamine [binding chondroitin sulfate (CS)chains] and aoidic sugars which are attached to these core proteins viaa linking tetrasaccharide moiety. Three major classes of HS-carryingcore proteins are present in living endothelial cells: cellmembrane-spanning syndecans, GPI-linked glypicans, and a secretedperlecan core protein [Rosenberg et al, J. Clin. Invest. 99: 2062-2070(1997)]. While the perlecan and glypican classes carry and bear HSchains almost exclusively, the syndecan core proteins are capable ofcarrying both HS and CS chains extracellularly. The appearance ofspecific glycosaminoglycan chains (such as HS and/or CS) extracellularlyon protein cores is regulated both by the structure of the particularcore protein as well as via the function of the Golgi apparatusintracellularly in a cell-type specific manner [Shworak et al., J. Biol.Chem. 26: 21204-21214 (1994)].

The syndecan class is composed of four closely related family proteins(syndecan-1,-2,-3 and -4 respectively) coded for by four different genesin-vivo. Syndecans-1 and -4 are the most widely studied members of thisclass and show expression in a variety of different cell types includingepithelial, endothelial, and vascular smooth muscle cells, althoughexpression in quiescent tissues is at a fairly low level [Bernfield etal., Annu. Rev. Cell Biol. 8: 365-393 (1992); Kim et al., Mol. Biol.Cell 5: 797-805 (1994)]. Syndecan-2 (also known as fibroglycan) isexpressed at high levels in cultured lung and skin fibroblasts, althoughimmunocytochemically this core protein is barely detectable in mostadult tissues. However, syndecan-3 (also known as N-syndecan)demonstrates a much more limited pattern of expression, being largelyrestricted to peripheral nerves and central nervous system tissues(although high levels of expression are shown in the neonatal heart)[Carey et al., J. Cell Biol 117: 191-201 (1992)]. All members of thesyndecan class are capable of carrying both HS and CS chainsextracellularly, although most of syndecan-associated biological effects(including regulation of blood coagulation, cell adhesion, and signaltransduction) are largely thought to be due to the presence of HS chainscapable of binding growth factors, or cell adhesion receptors and otherbiologically active molecules [Rosenberg et al., J. Clin. Invest. 22:2062-2070 (1997)].

Curiously, however, very little is presently known about and relativelylittle research attention has been paid to the function of the syndecancore proteins in-situ. Syndecan-1 expression has been observed duringdevelopment suggesting a potential role in the epithelial organizationof the embryonic ectoderm and in differential axial patterning of theembryonic mesoderm, as well as in cell differentiation [Sutherland etal., Development 113: 339-351 (1991); Trautman et al., Development 111:213-220 (1991)]. Also, mesenchymal cell growth during toothorganogenesis is associated with transient induction of syndecan-1 geneexpression [Vainio et al., Dev. Biol. 147: 322-333 (1991)]. Furthermore,in adult living tissues, expression of syndecan-1 and syndecan-4proteoglycans increases within arterial smooth muscle cells afterballoon catheter injury [Nikkari et al., Am. J. Pathol. 144: 1348-1356(1994)]; in healing skin wounds [Gallo et al., Proc. Natl. Acad. Sci.USA 91: 11035-11039 (1994)]; and in the heart following myocardialinfarction [Li et al., Circ. Res. 81: 785-796 (1997)]. In the latterinstances, the presence of blood-derived macrophages appears necessaryfor the induction of syndecan-1 and 4 gene expression. However, theeffects of changes in syndecan expression on cell behavior are presentlynot well understood. For example, this core protein is believed tomediate bFGF binding and cell activity. Overexpression of syndecan-1 in3T3 cells led to inhibition of bFGF-induced growth [Mali et al., J.Biol. Chem, 268: 24215-24222 (1993)]; while in 293T cells,overexpression of syndecan-1 augmented serum-dependent growth [Numa etal., Cancer Res. 5: 4676-4680 (1995)]. Furthermore, syndecan-1overexpression showed increased inter-cellular adhesion in lymphoidcells [Lbakken et al., J. Cell Biol 132: 1209-1221 (1996)] while alsodecreasing the ability of B-lymphocytes to invade collagen gels[Libersbach, B. F. and R. D. Sanderson, J. Biol. Chem. M: 20013-20019(1994)]. These ostensibly contradictory findings have merely added tothe uncertainty and the disparity of knowledge regarding the effects ofsyndecan expression.

In comparison, the glypican core protein class is composed of fivemurine and human members and a Drosophila dally homologue [Rosenberg etal., J. Clin. Invest. 99: 2062-2070 (1997)]. Unlike syndecans, theglypican members are fully extracellular proteins attached to the cellmembrane via a GPI anchor. Only one member of the class, glypican-1, isexpressed in endothelial cells. Another unique feature of the glypicanclass of proteoglycans is that they carry substantially only heparansulfate (HS) chains [Aviezer et al., J. Biol. Chem. 269: 114-121(1994)]. Consequently, while little is presently known about thebiological function of glypicans, they appear able to stimulate FGFreceptor 1 occupancy by bFGF and appear able to promote biologicalactivity for several different FGF family members [Steinfeld et al., J.Cell Biol. 133: 405-416 (1996)].

Finally, perlecan is the third and last class of heparan sulfate(HS)-carrying core proteins. Perlecan is a secreted proteoglycan thatalso has been implicated in regulation of bFGF activity [Aviezer et al.,Mol. Cell Biol, 17: 1938-1946 (1997); Steinfeld et al., J. Cell Biol.133: 405-416 (1996)]. However, little is known regarding this basallamina proteoglycan beyond its interaction with basic fibroblast growthfactor receptor.

In sum therefore, it is evident that the quantity and quality ofknowledge presently available regarding glycoseaminoglycan (GAG) bindingcore proteins is factually incomplete, often presumptive, and in someinstance apparently contradictory. Clearly the rule of specificproteoglycans as mediators under varying conditions is recognized;nevertheless, the mechanisms of action and the functional activity ofthe various individual classes of core proteins yet remains to beelucidated in full. Thus, while the role of proteoglycans in some mannerrelates to angiogenesis, there is no evidence or data known to datewhich clearly establishes the true functional value of proteoglycans norwhich establishes a use for proteoglycans as a means for stimulatingangiogenesis in-situ.

SUMMARY OF THE INVENTION

The present invention has multiple aspects and is definable in multiplecontexts. A first primary aspect and definition provides a prepared DNAsegment for placement in a suitable expression vector and transfectionof endothelial cells in-situ such that overexpression of extracellularmatrix heparan sulfate proteoglycan entities subsequently occursin-situ, said prepared DNA segment comprising:

-   -   at least one first DNA sequence coding for the extracellular        domain of a discrete proteoglycan entity that is expressed by a        transfected endothelial cell in-situ, said extracellular domain        first DNA sequence specifying the extracellular N-terminal        portion of an expressed proteoglycan entity which is then        located at and extends from the endothelial cell surface and is        capable of binding heparan sulfates to form an extracellular        matrix in-situ.    -   at least one second DNA sequence coding for the transmembrane        domain of a discrete proteoglycan entity that is expressed by a        transfected endothelial cell in-situ, said transmembrane domain        second DNA sequence specifying the medial portion of an        expressed proteoglycan entity which is then located at and        extends through the endothelial cell membrane and is joined with        said extracellular N-terminal portion of said expressed        proteoglycan entity; and    -   at least one third DNA sequence coding for the cytoplasmic        domain of the syndecan-4 molecule in said discrete proteoglycan        entity that is expressed by a transfected endothelial cell        in-situ, said syndecan-4 cytoplasmic domain third DNA sequence        specifying the cytoplasmic portion of an expressed proteoglycan        entity which is then present within the cytoplasm of a        transfected endothelial cell and is joined to said transmembrane        portion and said extracellular N-terminal portion of said        expressed proteoglycan entity.

A second primary aspect and definition provides a constructed expressionvector for transfection of endothelial cells in-situ such thatoverexpression of extracellular matrix haparan sulfate proteoglycanentities subsequently occurs in-situ, said constructed expression vectorcomprising:

-   -   a prepared DNA segment comprised of        -   (i) at least one first DNA sequence coding for the            extracellular domain of a discrete proteoglycan entity that            is expressed by a transfected endothelial cell in-situ, said            extracellular domain first DNA sequence specifying the            extracellular N-terminal portion of an expressed            proteoglycan entity which is then located at and extends            from the endothelial cell surface and is capable of binding            heparan sulfates to form an extracellular matrix in-situ,        -   (ii) at least one second DNA sequence coding for the            transmembrane domain of a discrete proteoglycan entity that            is expressed by a transfected endothelial cell in-situ, said            transmembrane domain second DNA sequence specifying the            medial portion of an expressed proteoglycan entity which is            then located at and extends through the endothelial cell            membrane and is joined with said extracellular N-terminal            portion of said expressed proteoglycan entity, and        -   (iii) at least one third DNA sequence coding for the            cytoplasmic domain of the syndecan-4 molecule in said            discrete proteoglycan entity that is expressed by a            transfected endothelial cell in-situ, said syndecan-4            cytoplasmic domain third DNA sequence specifying the            cytoplasmic portion of an expressed proteoglycan entity            which is then present within the cytoplasm of a transfected            endothelial cell and is joined to said transmembrane portion            and said extracellular N-terminal portion of said expressed            proteoglycan entity; and an expression vector carrying said            prepared DNA segment and suitable for transfection of            endothelial cells in-situ.

A third primary aspect and definition provides an in-situ transfectedendothelial cell which overexpresses extracellular matrix heparansulfate proteoglycans and positions on the proteoglycans at the cellsurface, said in-situ transfected endothelial cell comprising:

-   -   a viable endothelial cell previously transfected in-situ with a        constructed expression vector such that said transfected        endothelial cell overexpresses discrete extracellular matrix        heparan sulfate proteoglycan entities coded for by said vector,        said overexpressed proteoglycan entities being comprised of        -   (i) an extracellular N-terminal portion which is located at            and extends from the transfected endothelial cell surface            and which binds heparan sulfates to form an extracellular            matrix in-situ, said extracellular N-terminal portion being            the expressed product of at least one first DNA sequence in            the constructed expression vector coding for the            extracellular domain of said proteoglycan entity expressed            by the transfected endothelial cell in-situ,        -   (ii) a transmembrane medial portion which is located at and            extends through the endothelial cell membrane and is joined            with said extracellular N-terminal portion of said            proteoglycan entity, said transmembrane medial portion being            the expressed product of at least one second DNA sequence in            the constructed expression vector coding for the            transmembrane domain of said proteoglycan entity expressed            by the transfected endothelial cell in-situ, and        -   (iii) a syndecan-4 cytoplasmic portion present within the            cytoplasm of the transfected endothelial cell which is            joined to said transmembrane portion and said extracellular            N-terminal portion of said proteoglycan entity, said            syndecan-4 cytoplasmic portion being the expressed product            of at least one third DNA sequence in the constructed            expression vector coding for the cytoplasmic domain of the            syndecan-4 molecule of said proteoglycan entity expressed by            the transfected endothelial cell in-situ.

A fourth primary aspect, and definition provides a method forstimulating angiogenesis in-situ within a living tissue comprisingvascular endothelial cells, said method comprising the steps of:

-   -   transfecting vascular endothelial cells within a living tissue        with a constructed expression vector such that the resulting        transfected vascular endothelial cells overexpress discrete        extracellular matrix heparan sulfate proteoglycan entities coded        for by said constructed expression vector, said overexpressed        proteoglycan entities being comprised of        -   (i) an extracellular N-terminal portion which is located at            and extends from the transfected vascular endothelial cell            surface and binds heparan sulfates to form an extracellular            matrix in-situ, said extracellular N-terminal portion being            the expressed product of at least one first DNA sequence in            the constructed expression vector coding for the            extracellular domain of said proteoglycan entity expressed            by a transfected vascular endothelial cell in-situ,        -   (ii) a transmembrane medial portion which is located at and            extends through a transfected vascular endothelial cell            membrane and is joined with said extracellular N-terminal            portion of said proteoglycan entity, said transmembrane            medial portion being the expressed product of at least one            second DNA sequence in the constructed expression vector            coding for the transmembrane domain of said proteoglycan            entity expressed by a transfected vascular endothelial cell            in-situ, and        -   (iii) a syndecan-4 cytoplasmic portion present within the            cytoplasm of a transfected vascular endothelial cell which            is joined to said transmembrane portion and said            extracellular N-terminal portion of said expressed            proteoglycan entity, said syndecan-4 cytoplasmic portion            being the expressed product of at least one third DNA            sequence in the constructed expression vector coding for the            cytoplasmic domain of the syndecan-4 molecule of said            proteoglycan entity expressed by a transfected vascular            endothelial cell in-situ; and    -   allowing said transfected vascular endothelial cells bearing        said overexpressed extracellular matrix proteoglycan entities to        stimulate angiogenesis in-situ.

BRIEF DESCRIPTION OF THE FIGURES

The present invention can be more easily understood and betterappreciated when taken in conjunction with the accompanying drawing, inwhich:

FIG. 1 is a representation of a prepared DNA sequence fragment;

FIG. 2 is a recitation of the DNA sequence coding for the extracellulardomain of syndecan-1[SEQ ID NO: 1];

FIG. 3 is a recitation of the DNA sequence coding for extracellulardomain of syndecan-2 [SEQ ID NOS:2 & 3];

FIG. 4 is a recitation of the DNA sequence coding for the extracellulardomain of syndecan-3 [SEQ ID NO:4];

FIG. 5 is a recitation of the DNA sequence coding for the extracellulardomain of syndecan-4 [SEQ ID NO:5];

FIG. 6 is a recitation of the DNA sequence coding for the extracellulardomain of glypican-1[SEQ ID NOS:6 & 7];

FIG. 7 is a recitation of the DNA sequence coding for the transmembranedomain of syndecan-1[SEQ ID NO:8];

FIG. 8 is a recitation of the DNA sequence coding for the transmembranedomain of syndecan-2 [SEQ ID NOS:9 & 10];

FIG. 9 is a recitation of the DNA sequence coding for the transmembranedomain of syndecan-3 [SEQ ID NO:11];

FIG. 10 is a recitation of the DNA sequence coding for the transmembranedomain of syndecan-4 [SEQ ID NO:12];

FIG. 11 is a recitation of the DNA sequence coding for the transmembranedomain of GP1 [SEQ ID NOS:13 & 14];

FIG. 12 is a recitation of the DNA sequence coding for the transmembranedomain of perlecan [SEQ ID NO:15];

FIG. 13 is a recitation of the DNA sequence coding for the cytoplasmicdomain of syndecan-4 [SEQ ID NO:16];

FIG. 14 is a graph illustrating the in-vitro growth assays ofECV-derived cell clones;

FIGS. 15A-15C are photographs showing the results of Matrigel growthsassays;

FIG. 16 is a graph illustrating the effect of syndecan constructexpression on endothelial cell migration in Boyden chamber assays;

FIGS. 17A-17F are photographs showing BudR uptake in opiop homozygous(−1−) and heterozygous (+1−) mice;

FIG. 18 is a photograph showing Northern blot analysis of geneexpression in PR-39 transgenic mice; and

FIG. 19 is a graph illustrating in-vitro microvascular reactivity inPR-39 transgenic mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides both the tangible means and the methodsfor causing an overexpression of extracellular, heparan sulfatecarrying, proteoglycans on-demand at and through the surface ofendothelial cells; and via such on-demand overexpression ofproteoglycans to stimulate angiogenesis in-situ. The tangible meansinclude a prepared DNA segment comprising sequences coding for anextracellular domain, a transmembrane domain, and the cytoplasmic domainof the syndecan-4 protein; as well as a constructed expression vectorfor the transfection of endothelial cells in-situ such thatoverexpression of extracellular matrix, heparan sulfate bearing,proteoglycan entities subsequently occurs in-situ. The resultingtransfected endothelial cell overexpresses proteoglycans and positionsthem at the cell surface —thereby providing the structural andfunctional entities by which to stimulate angiogenesis in-situ.

A number of major benefits and advantages are therefore provided by themeans and methods comprising the present invention. These include thefollowing:

-   1. The present invention provides in-situ stimulation for    angiogenesis. By definition, therefore, both in-vivo and in-vitro    circumstances of use and application are envisioned and expected.    Moreover, the endothelial cells which are to be transfected such    that overexpression of proteoglycans subsequently occurs, may be    alternatively isolated endothelial cells, be part of living tissues    comprising a variety of other cells such as fibrocytes and muscle    cells, and may also comprise part of specific organs in the body of    a living human or animal subject. While the user shall choose the    specific conditions and circumstances for practicing the present    invention, the intended scope of application and the envisioned    utility of the means and methods described herein apply broadly to    living cells, living tissues, functional organs and systems, as well    as the complete living body unit as a viable whole.-   2. The present invention has a variety of different applications and    uses. Of clinical and medical interest and value, the present    invention provides the opportunity to stimulate angiogenesis in    tissues and organs in a living subject which has suffered defects or    has undergone anoxia or infarction. A common clinical instance is    the myocardial infarction or chronic myocardial ischemia of heart    tissue in various zones or areas of a living human subject. The    present invention thus provides opportunity and means for specific    site stimulation and inducement of angiogenesis under controlled    conditions. The present invention also has major research value for    research investigators in furthering the quality and quantity of    knowledge regarding the mechanisms controlling angiogenesis under a    variety of different conditions and circumstances.-   3. The present invention envisions and permits a diverse range of    routes of administration and delivery means for introducing a    constructed expression vector to a specific location, site, tissue,    organ, or system in the living body. A variety of different    expression vectors are available to the practitioner; and a diverse    and useful range of delivery systems which are conventionally    available and in accordance with good medical practice are adapted    directly for use. In this manner, not only are the means for    transfection under the control of the user, but also the manner of    application and limiting the locale or area of intentional    transfection of endothelial cells can be chosen and controlled.-   4. The user also has the choice and discretion of the manner in    which the DNA segment is prepared —so long as the prepared DNA    fragment conforms to the minimal requirements set forth herein.    Thus, the prepared DNA sequence fragment may comprise the entire    syndecan-4 DNA sequence in each of the required extracellular,    transmembrane, and cytoplasmic domains. However, it is expected and    envisioned that the more frequent choice will be a chimera core    protein structure which comprises only the syndecan-4 cytoplasmic    domain but incorporates transmembrane and extracellular domains    which are not native to the DNA of syndecan-4. Thus, the majority of    prepared DNA sequenced fragments will be chimeric DNA segments    ligated together intentionally using recombinant techniques and    methods to form a unitary DNA fragment.-   5. The present invention provides a unique capability and control    for stimulating angiogenesis in-situ by genetic manipulation of the    endothelial cells as they exist within the tissues and organs as    found. This level of gene control and utilization of the expression    mechanisms found within the cytoplasms of the endothelial cells    themselves provides a point of intentional intervention which    harnesses and utilizes the cellular systems of the endothelial cells    themselves to produce the intended and desired result. The    transfected endothelial cells in-situ are thus minimally altered    from their original genetic constituents; and the methodology    utilizes the natural regulatory and protein producing systems of the    endothelial cells themselves to provide the overexpression of    proteoglycans which are located and positioned in the normally    expected manner by the endothelial cells as part of the normal    homeostatic mechanisms.

Accordingly, by the very requirements of the present invention it isthus important, if not essential, that the user be at least familiarwith the many techniques for manipulating and modifying nucleotides andDNA fragments which have been reported and are today widespread in useand application. Merely exemplifying the many authoritative texts andpublished articles presently available in the literature regardinggenes, DNA nucleotide manipulation and the expression of proteins frommanipulated DNA fragments are the following: Gene Probes for Bacteria(Macario and De Marcario, editors) Academic Press Inc., 1990; GeneticAnalysis, Principles Scope and Objectives by John R. S. Ficham,Blackwell Science Ltd., 1994; Recombinant DNA Methodology II (Ray Wu,editor), Academic Press, 1995; Molecular Cloning, A Laboratory Manual(Maniatis, Fritsch, and Sambrook, editors), Cold Spring HarborLaboratory, 1982; PCR (Polymerase Chain Reaction), (Newton and Graham,editors), Bios Scientific Publishers, 1994; and the many referencesindividually cited within each of these publications. All of thesepublished texts are expressly incorporated by reference herein.

In addition, a number of issued U.S. Patents and published patentapplications have been issued which describe much of the underlying DNAtechnology and many of the conventional recombinant practices andtechniques for preparing DNA sequences coding for core proteins such assyndecan-4. Merely exemplifying some of the relevant patent literaturefor this subject are: U.S. Pat. Nos. 5,486,599; 5,422,243; 5,654,273;4,356,270; 4,331,901; 4,273,875; 4,304,863; 4,419,450; 4,362,867;4,403,036; 4,363,877; as well as Publications Nos. WO9534316-A1;WO9412162-A1; WO9305167-A1; WO9012033-A1; WO9500633; WO9412162; andR09012033. All of these patent literature publications are alsoexpressly incorporated by reference herein.

I. Constructed Core Protein DNA Fragments

A primary component part of the subject matter as a whole comprising thepresent invention is the manufacture and proper use of a prepared DNAsegment intended for placement in a suitable expression vector; anduseful for transfection of endothelial cells in-situ, under both in-vivoand in-vitro conditions, such that overexpression of extracellularmatrix heparan sulfate carrying proteoglycans subsequently occursin-situ. The prepared DNA segment is a manufactured or synthesizednucleotide fragment which preferably exists as a single, coiled strandof DNA bases in series; and constitutes sufficient DNA information tocode for three requisite domains as illustrated by FIG. 1.

FIG. 1 is a simplistic and broadly representational illustration of theprepared DNA fragment after manufacture or synthesis. As seen therein,the prepared DNA segment comprises at least a first DNA sequence codingfor the extracellular domain of a discrete and identifiable proteoglycanentity which, after being expressed by a transfected endothelial cellin-situ, yields a specified N-terminal portion of an expressedproteoglycan entity. This N-terminal portion is the extracellular regionof the expressed proteoglycan molecule which is then located at andextends from the transfected endothelial cell surface. This extended,extracellular N-terminal region (expressed as specific amino acidresidues in sequence) is capable of binding heparan sulfates at the cellsurface thereby forming an extracellular heparan sulfate matrix in-situ.

The prepared DNA segment fragment illustrated by FIG. 1 must alsoprovide at least one se on d DNA sequence coding for the transmembranedomain of a discrete proteoglycan entity that is expressed by atransfected endothelial cell in-situ. This transmembrane domain secondDNA sequence codes for and specifies the amino acid residue sequence ofthe medial or central portion of an expressed proteoglycan entity by thetransfected endothelial cell. The medial portion or central region ofthe expressed proteoglycan is located at and extends through theendothelial cell membrane and is directly joined with and to theextracellular N-terminal portion of the expressed proteoglycan thenextending from the cell surface.

The final requisite component of the prepared DNA segment illustrated byFIG. 1 comprises at least one third DNA sequence coding for thecytoplasmic domain of the syndecan 4 molecule within the discreteproteoglycan entity that is expressed by a transfected endothelial cellin-situ. This third DNA sequence specifies the cytoplasmic domain of thesyndecan-4 DNA; and thus requires the expression of the particular aminoacid residues which identify the syndecan-4 cytoplasmic region of thesyndecan-4 core protein structure. While some small variation ispermitted within the third DNA sequence specifying the cytoplasmicdomain of the syndecan-4 amino acid structure, it is essential andrequired in every embodiment of the prepared DNA fragment which is thepresent invention that the expressed cytoplasmic region of theproteoglycan entity then present within the cytoplasm of a transfectedendothelial cell be identifiably recognized as being a syndecan-4 aminoacid residue type. In addition, the expressed cytoplasmic portionconstituting the syndecan-4 amino acid sequence must be present withinthe cytoplasm of a transfected endothelial cell; and be joined to thetransmembrane portion and the extracellular N-terminal portion of theexpressed proteoglycan entity.

The Heterogeneous Domains Joined Together as a Unitary Fragment

It will be recognized and appreciated that the prepared DNA sequence isintended to be primarily, but not always, a heterogeneous DNA structurewhich joins together individual and separate DNA sequences as a unitaryfragment. The cytoplasmic domain constituting the third DNA sequence ofthe prepared fragment is limited and restricted to those DNA bases insequence which recognizably and identifiably code for the syndecan-4amino acid residues. Although single point or small variant alternationsor modifications in the DNA base sequence is permissible and expected,the overall domain must be in each and every instance recognizable andidentifiable (using appropriate analytical means) as representative ofthe cytoplasmic region of the syndecan-4 molecular structure.

In comparison, the practitioner or intended user has the choice of manydifferent DNA sequences and formats when choosing and selecting DNAsequences coding for the extracellular domain coding for the N-terminalregion and the transmembrane domain coding for the central or medialregion of the proteoglycan molecule to be expressed. Thus, the user mayconstruct the entirety of the syndecan-4 DNA base sequence in itsentirety such that a complete syndecan-4 core protein is subsequentlyexpressed by a transfected endothelial cell. However, it is expectedthat in many instances the heterogeneous combination of individual andseparate DNA base sequences representative of other and different coreprotein structures will be utilized; and that the resulting expressedproteoglycan entity will therefore be a chimeric core protein havingdifferent amino acid residues constituting the transmembrane region andthe extracellular region of the expressed proteoglycan entity. Thus itis expected and envisioned that the first DNA sequence may be the DNAcoding for the glypican-1 amino acid residues; while the second DNAsequence coding for the transmembrane domain may be representative ofthe syndecan-1 amino acid structure. Thus, the availability and use ofheterogeneous prepared DNA fragments linking together first, second, andthird DNA sequences —each of which is representative of a different coreprotein content and structure —thus will yield the expression of achimeric proteoglycan entity which does not and cannot occur in nature.

In addition, the present availability of manufacturing heterogeneous DNAfragments which will yield an expressed chimera core protein in atransfected endothelial cell in-situ allows the intended user to chooseand more carefully align the amino acid composition of the expressedproteoglycan entity to be in accordance with and more compatible to theparticular clinical problem and specific living tissue which is theintended treatment target. Thus, if damaged myocardium is the tissueintended as the target for treatment, the manufacture of theheterogeneous fragment might include an extracellular domain (the firstDNA sequence) coding for the glycipan-1 region; which is joined to thetransmembrane DNA domain (the second DNA sequence) which itself codesfor a syndecan-2 amino acid region; which in turn is linked to thecytoplasmic domain (the third DNA sequence) which must code for thesyndecan-4 region. In comparison, however, if the targeted tissue islung tissue, the extracellular domain might be representative of thesyndecan-1 amino acid region; while the transmembrane domain representsthe DNA coding for the amino acids of the syndecan-3 region; and thecytoplasmic domain continues to code exclusively for the syndecan-4region. In other words, the extracellular domain can be specificallytailored to an environment where it will be expressed.

In this manner, the manufacturer or intended user may customize andtailor the DNA sequences constituting the extracellular domain and/orthe transmembrane domain as far as possible to best meet or suit theparticular tissue, clinical condition, or pathology then existing andcritical to the particular application of interest. The range andvariety of choices, therefore, allows the manufacturer and intended usera greater degree of flexibility, of potential therapeutic effects, and agreater degree of individuality than has ever been possible before thepresent invention was made.

Manufacture of the Prepared DNA Sequence Fragment

It is expected and intended that the conventionally known and commonlyused recombinant DNA materials, procedures, and instrumentation will beemployed for the manufacture of the prepared DNA sequence fragments.Thus, the entire prepared DNA sequence structure including the entiretyof the extracellular domain and the transmembrane domain, and thecytoplasmic domain coding for the syndecan-4 structure may besynthesized directly from individual bases using the commerciallyavailable instruments and techniques. Alternatively, the DNA sequencesexisting in naturally occurring core proteins may be replicated; and thecDNA isolated from individual clones using the appropriate enzymes andprotocols. Regardless of the methods and means of manufacture, any andall of these protocols, procedures, systems, or instruments which willyield the prepared DNA sequence as an discrete fragment is suitable andappropriate for use with the present invention.

A preferred technique, procedure, and methodology for preparing the DNAfragment as a whole is given in the Materials and Methods portion of theExperiments presented hereinafter. The described method, however, ismerely one among many conventionally known and available for thispurpose.

A. The Extracellular Domain DNA Sequence

The manufacturer or user has a substantial choice in the range andvariety of the DNA sequences suitable for use as the extracellulardomain. A representative, but non-exhaustive, listing of suitablechoices is provided by Table 1 below.

TABLE 1 Representative Extracellular Domain DNA Sequence FragmentsExtracellular Domain DNA Sequence Type Variant Recited By syndecan-1FIG. 2 [SEQ ID NO:1] syndecan-2 FIG. 3 [SEQ ID NO:2] syndecan-3 FIG. 4[SEQ ID NO:4] syndecan 4 FIG. 5 [SEQ ID NO:5] glypican-1 FIG. 6 [SEQ IDNO:6]B. The Transmembrane Domain DNA Sequences

The manufacturer or user also has substantial choice in the range andvariety of the DNA sequences to be used as the transmembrane domainsequence coding for the medial or central region of the expressedproteoglycan entity. A representative, but non-exhaustive, listing ofthe second DNA sequence in the prepared fragment constituting and codingfor the transmembrane domain is provided by Table 2 below.

TABLE 2 Representative Transmembrane Domain DNA Sequence FragmentsTransmembrane Domain DNA Sequence Type Variant Recited By syndecan-1FIG. 7 [SEQ ID NO:8] syndecan-2 FIG. 8 [SEQ ID NO:9] syndecan-3 FIG. 9[SEQ ID NO:11] syndecan 4 FIG. 10 [SEQ ID NO:12] GPI FIG. 11 [SEQ IDNO:13] perlecan FIG. 12 [SEQ ID NO:14]C. The Cytoplasmic Domain Coding For The Syndecan-4 Peptide

The third requisite cytoplasmic domain must code for the amino acidresidue structure representative of the syndecan-4 core protein. Asshown experimentally by the data presented hereinafter, only thesyndecan-4 cytoplasmic region and peptide structure allows forfunctional stimulation of angiogenesis in situ. For this reason, it isessential and required in each embodiment of the present invention thatthe third DNA sequence coding for the cytoplasmic domain in theexpressed proteoglycan entity in a transfected endothelial cell berepresentative of and analytically identifiable as the syndecan-4 aminoacid residue structure. A representative recitation of the DNAconstituting the cytoplasmic domain of the syndecan-4 molecule ispresented by FIG. 13 herein.

It will be noted and recognized that very little variability andsubstitution within the specific DNA base sequencing of the cytoplasmicdomain of the syndecan-4 molecule is permitted. While some changes areexpected, be they point mutations, block substitutions and the like, theexpected or envisioned degree of variability which might be present orpermitted for the cytoplasmic domain DNA is believed to be quitelimited.

As representative examples: The last four amino acids (EFYA) [SEQ IDNO:25] cannot be changed or modified. Similarly, regarding the Serineresidue at position 181: a mutation to an Alanine residue potentiatesactivation; while a mutation to Glutamate inhibits cell growth in adominant fashion (dominant-negative mutation). Finally, the LGKKPIYKKsequences [SEQ ID NO:24] probably cannot be altered at all.

Expression Vectors And Means For Delivery In-Situ

A variety of methods are conventionally known and presently available tothe user or practitioner of the present invention in order to introduceand deliver a prepared DNA sequence fragment to the intended targetin-situ. The means for delivery envision and include in-vivocircumstances; ex-vivo specimens and conditions; and in-vitro culturecircumstances. In addition, the present invention intends and expectsthat the use of the prepared DNA sequence fragment in a suitableexpression vector and route of administration will be delivered toliving tissues comprising endothelial cells, and typically vascularendothelial cells which constitute the basal layer of cells incapillaries and blood vessels generally. Clearly, the cells themselvesare thus eukarytoic, typically mammalian cells from human and animalorigin; and most typically would include the higher order mammals suchas humans and domesticated animals kept as pets or sources of foodintended for consumption. Accordingly, the range of animals includes alldomesticated varieties involved in nutrition including cattle, sheep,pigs and the like; as well as those animals typically used as pets orraised for commercial purposes including horses, dogs, cats, and otherliving mammals typically living with and around humans.

Clearly, the expression vectors then must be suitable for transfectionof endothelial cells in living tissues of mammalian origin and thus becompatible with that type and condition of cells under both in-vivoand/or in-vitro conditions. The expression vectors thus typicallyinclude plasmids and viruses as expression vectors.

The range and variety of plasmids suitable for use with the presentinvention are broadly available and conventionally known in thetechnical and scientific literature. A representative, butnon-exhaustive, listing is provided by Table 3 below.

TABLE 3 Preferred Mammalian Plasmid Expression Vectors Plasmid VectorspHβ-APr-1-neo EBO-pcD-XN pcDNAI/amp pcDNAI/neo pRc/CMV pSV2gpt pSV2neopSV2-dhfr pTk2 pRSV-neo pMSG pSVT7 pKo-neo pHyg

Alternatively, a wide and divergent variety of viral expression vectorssuitable for insertion of the prepared DNA sequence fragment andsubsequent transfection of endothelial cells in-situ is conventionallyknown and commonly available in this field. The particular choice ofviral vector and the preparation of the fully constructed expressionvector incorporating the prepared DNA sequence fragment is clearly amatter of personal convenience and choice to the intended manufactureror user; but should be selected with a eye towards the intendedapplication and the nature of the tissues which are the intended target.A representative, but non-exhaustive, listing of preferred viralexpression vectors suitable for use as constructed vectors bearing theprepared DNA sequence fragment is provided by Table 4 below.

TABLE 4 Preferred Viral Expression Vectors Bovine papilloma virus(BPV-1); Epstein-Barr virus (phEBO; pREP-derived, and p205); Retrovirus;Adenovirus; AAV (adeno-associated virus) Lentivirus

Clearly, both the plasmid based vectors and the viral expression vectorsconstitute means and methods of delivery which are conventionallyrecognized today as “gene therapy” modes of delivery. However, thisoverall approach is not the only means and method of delivery availablefor the present invention.

Injection of Recombinant Proteins

Intracoronary delivery is accomplished using catheter-based deliveriesof recombinant human protein dissolved in a suitable buffer (such assaline) which can be injected locally (i.e., by injecting into themyocardium through the vessel wall) in the coronary artery using asuitable local delivery catheter such as a 10 mm InfusaSleeve catheter(Local Med, Palo Alto, Calif.) loaded over a 3.0 mm×20 mm angioplastyballoon, delivered over a 0.014 inch angioplasty guidewire. Delivery wasaccomplished by first inflating the angioplasty balloon to 30 psi, andthen deliverying the protein through the local delivery catheter at 80psi over 30 seconds (this can be modified to suit the deliverycatheter).

Intracoronary bolus infusion can be accomplished by a manual injectionof the protein through an Ultrafuse-X dual lumen catheter (SciMed,Minneapolis, Minn.) or another suitable device into proximal orifices ofcoronary arteries over 10 minutes.

Pericardial delivery is accomplished by instillation of theprotein-containing solution into the pericardial sac. The pericardium isaccessed either via a right atrial puncture, transthoracic puncture orvia a direct surgical approach. Once the access is established, thematerial is infused into the pericardial cavity and the catheter iswithdrawn. Alternatively, the delivery is accomplished usingslow-release polymers such as heparin-alginate or ehylene vinyl acetate(EVAc). In both cases, once the protein is integrated into the polymer,the desired amount of polymer is inserted under the epicardial fat orsecured to the myocardial surface using, for example, sutures. Inaddition, polymer can be positioned along the adventitial surface ofcoronary vessels.

Intramyocardial delivery can be accomplished either under direct visionfollowing thoracotomy or using thoracoscope or via a catheter. In eithercase, the protein containing solution is injected using a syringe orother suitable device directly into the myocardium. Up to 2 cc of volumecan be injected into any given spot and multiple locations (up to 30injections) can be done in each patient. Catheter-based injections arecarried out under fluoroscopic, ultrasound or Biosense NOGA guidance. Inall cases after catheter introduction into the left ventricle thedesired area of the myocardium is injected using a catheter that allowsfor controlled local delivery of the material.

III. Examplary Applications and Preferred Routes Of Administration

A variety of approaches, routes of administration, and delivery methodsare available using the constructed expression vector comprising aninserted DNA sequence fragment coding for a proteoglycan entity. Amajority of the approaches and routes of administration describedhereinafter are medical applications and specific clinical approachesintended for use with human patients having specific medical problemsand pathologies. It is expected that the reader is familiar generallywith the typical clinical human problem, pathology, and medicalconditions described herein; and therefore will be able to follow andeasily understand the nature of the intervention clinically using thepresent invention and the intended outcome and result of the clinicaltreatment —particularly as pertains to the stimulation of angiogenesisunder in-vivo treatment conditions. A representative listing ofpreferred approaches is given by Table 5 below.

TABLE 5 Preferred Routes Of Administration Catheter-based(intracoronary) injections and infusions; Direct myocardial injection(intramyocardial guided); Direct myocardial injection (directvision-epicardial-open chest or under thorascope guidance); Localintravascular delivery; Liposome-based delivery; Delivery in associationwith “homing” peptides.

Experimental and Empirical Data

To demonstrate the merits and value of the present invention, a seriesof planned experiments and empirical data are presented below. It willbe expressly understood, however, that the experiments described and theresults provided are merely the best evidence of the subject matter as awhole which is the invention; and that the empirical data, while limitedin content, is only illustrative of the scope of the inventionenvisioned and claimed.

A. Materials and Methods:

Expression Constructs and Cell Culture

Immortalized ECV304 cells (ATCC, Bethesda, Md.) were cultured inDulbecco's modified Eagle's medium (DMEM, Gibco-BRL) supplemented withheat-inactivated 10% fetal bovine serum (FBS, Gibco-BRL), 2 mMglutamine, 100 U/ml penicillin and 100 μg/ml streptomycin at 37° C. in5% Co₂. Full length coding region cDNAs for rat syndecan-4 and ratglypican-1 expression constructs were prepared in a retroviral vectorMSCV2.2 by cloning a BamHI/Hpa1 fragment of rat syndecan-4 into cDNAinto BgLII/Eco Hpa1 fragment vector and BamH1/EcOR1 fragment of ratglypican-1 into BgLII/Eco R1 sites of the same vector. Syndecan/glypicanchimeras were created via PCR mutagenesis; cloned into the pcDNA3;sequence d; and shuttled into the MSCV2.2 vector. The syndecan-4-GPI(S4-GPI) construct was created by deleting the C-terminal end of ratsyndecan-4 sequence starting With ²⁴⁷ Gln and replacing it with theC-terminal sequence of rat glypican-1 starting with ⁵¹⁰Ser. Theglypican-syndecan-4 cytoplasmic domain (G1-S4c) construct was created byreplacing C-terminal sequence of rat glypican-1 starting with ⁵¹⁰Serwith amino acids 247-321 of the rat syndecan-4 sequence. The createdchimera thus contains both transmembrane and cytoplasmic regions ofsyndecan-4. Transfection of the MSCV2.2 vector alone was used togenerate a control ECV cell population.

Retroviral transduction

The virus for transductions was produced by calcium phosphate transienttransfection (29) of 10 μg of each construct on amphotropic Phoenixpackaging cells (ATCC). Viral supernatants were collected after 36, 48and 72 hrs, sterile filtered through 0.2 μm filter and then transferredto ECV-304 cells at 32° C. in the presence of 25 μg/ml DEAE-dextran.Typical viral titers in the supernatant were approximately 6−8×10⁵infectious particles/ml. Virus exposure was repeat ed 4 times for eachconstruct; following the last exposure the cells were cultured in 10%FBS-DMEM supplemented with 400 μg/ml active G418 (Sigma) for two weeks.

Growth and Migration Assays

For growth assays, 100,000 cells were plated in 6 well cell cultureplates and allowed to attach overnight. At that time, the cells werewashed 3 times with phosphate-buffered saline (PBS) and the medium waschanged to DMEM supplemented with 0.25% FBS. Twenty four hours later, 25ng/ml of bFGF (Chiron Corp.) were added to the cell culture medium. Cellcounts were then obtained at 24 hr intervals starting with the time ofexposure to bFGF by trypsinizing the well and counting cell suspensionson a Coulter counter (Coulter Corp.).

Migration assays were carried out using modified Boyden chambers(Neuroprobe, Inc.). ECV 304 cells and derived clones were grown in 10%FBS-DMEM supplemented with 5 ng/ml DiI (DiIC₁₈;1,1-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanide perchlorate,Molecular Probes) living cell fluorescent stain overnight. Followingthat, the cells were trypsinized, washed with DMEM, diluted in DMEMsupplemented with 0.5% FBS and seeded in wells at 60,000 cells per well.The cell containing compartments were separated from the lower wells by25×80 mm polycarbonate filters with 8 μm pores (Poretics Corp.). Thelower chambers were filled with 0.5% FBS-DMEM supplemented with 50 ng/mlbFGF and the entire apparatus was incubated in a tissue cultureincubator at 37° C., 5% CO₂ for 4.5 hours. After that time non-migratingcells were removed by washing the upper wells with PBS, the uppersurfaces of the filters were scraped with a plastic blade, and thefilters were fixed in 4% formaldehyde for 1 min and placed on a glassslide. The migrated cells were imaged using a digital SesSys cameraattached to a Nikon fluorescent microscope. For each slide, 3non-overlapping lower power (5×) fields were selected for analysis.Following image acquisition using PMIS image processing software(Photometrics, Ltd.) the number of cells was automatically determinedusing Optimas 6.0 software (Bioscan, Inc.).

Matrigel Growth Assay

Growth factor depleted Matrigel (Becton Dickinson) plates were preparedby adding 0.5 ml of thawed Matrigel to a well of refrigerated 24 welltissue culture plate. The gel was allowed to solidify for one hour at37° C. and overlaid with 1 ml of 0.5% FBS-DMEM containing 30,000 cells.The cell culture was carried out at 37° C. in a humidified atmospheresupplemented with 5% CO₂. The analysis of cell growth was carried out byobtaining lower (10×) and high (40×) power images of the wells with adigital SesSys camera focused on the surface of the gel using aninverted Nikon fluorescent microscope. The cell-free area was thedetermined using Optimas 6.0 software.

RNA Isolation and RT PCR Analysis

For RNA analysis of syndecan-4 and PR-39 expression, cell cultures weretrypsinized, pelleted, and total RNA was prepared using TRI Reagent(Sigma Biosciences). The RNA pellet was dissolved in RNase-free waterand ethanol precipitated. For RT-PCR analysis, 0.2 μg total RNA wereused for reverse transcription with a 15 pmol of oligo(dT)₂₀ primer, 75mM KCl, 3 mM MgCl₂, 10 mM DTT, 0.5 mM each dNTP in 50 mM Tris-HCl (pH8.3) buffer. The mixture was heated to 70° C. for 10 min, then cooled to37° C. while 1 μl of Super Script II reverse transcriptase (200 U/μl,Life Technologies, Inc.) was added. The reaction was allowed to proceedfor 1 hr at 37° C. and then terminated by heating for 5 min followed bychilling to 4° C. 1 μ1 of the RT reaction mixture was used for PCRamplification using specific primers. The PCR reaction was carried outin the presence of 1.5 mM MgCl₂, 0.2 mM dNTP, 400 nM 3′ and 5′ primersand 2.5 U of Taq DNA polymerase (Boehringer Mannheim, Inc.). Thefollowing specific primers were used: Glypican-1: 5′: CCC CGC CAG CAAGAG CCG GAG CT; [SEQ ID NO:18] 3′: GTG AGG CTC TGG GCG AGT GGG GG, [SEQID NO:19] Syndecan-4: 5′ (with Sac I restriction site): ATA GAG CTC TTGGAA CCA TGG CFC CTG TCT GCC; [SEQ ID NO:20] 3′: (with Eco RI restrictionsite): GGA ATT CCA GGT TTT ATT ATC TTT TTA TC [SEQ ID NO:21].

For standardization purposes a conserved region of human and mouseGAP-DH gene was chosen for amplification as a control template. Thefollowing primers were used: 5′: CGT ATT GGG CGC. CGT GTC ACC AGG GC;[SEQ ID NO:22] 3′: GGC CAT GAG CTC CAC CAC CCT GTT CG [SEQ ID NO:23].All reactions were carried out using GeneAmp PCR 2400 system (PerkinsElmer, Inc.) as follows: 94° C. (1 min), 50-55° C. (30 sec), 72° C. (1.5min). The additional final extension step was performed at 72° C. for 7min. A total of 30 cycles were done for each reaction. Following PCRamplification, reaction products were subjected to 1% agarose gelelectrophoresis and the amount of specific message was expressed as aratio to GAP-DH message.

Determination of Heparan Sulfate Mass in Cultured Cells

To determine the total mass of heparan sulfate chains, endothelial cellcultures were washed twice with PBS and incubated for 24 h with 2 mCi ofNa₂ ³⁵SO₄ in 2 ml of a modified basal Eagle medium supplemented with 1%Neutrodoma-SP. At the end of labeling, cells are washed with cold PBSand incubated with a lysis buffer followed by centrifugation at 15,000×gfor 10 min at 4° C. Total proteoglycans (PG) are isolated from thesupernatant by DEAE chromatography. Glycosaminoglycans were cleaved fromthe total PG pool by β-elimination and the relative content of HS and CSis determined by appropriate enzyme digests with chondroitinase ABC orFlavobactefium heparatinase 1 and 3. Preliminary experiments onmicrovascular endothelial cells demonstrated that the sum of HS and CSsulfate accounted for >98% of the total PG sulfate.

Scatchard Analysis of low Affinity BFGF Binding Sites

For determination of the number and affinity of bFGF heparan sulfatebinding sites, endothelial cells were grown to near confluence in 24well dishes in 10% FBS-DMEM. After two washes with cold PBS, 200 μl ofbinding buffer (25 mM HEPES, pH 7.4, 0.1% BSA, 0.05% gelatin in M199medium), 6×10⁶ cpm (0.5 ng/ml) ²⁵-1-bFGF (DuPont, specific activity 2000C/mmol), and increasing amounts (0-600 ng/ml) cold bFGF were added toeach well. The cells were incubated at 4° C. for 2 h with gentleagitation; at the end of that time, the cells were washed three timeswith 1 ml PBS containing 0.1% BSA and then incubated with 1% Triton-X100 in 5 ml water supplemented with 0.01% BSA (Sigma) for 30 min at roomtemperature with vigorous shaking. Following this, 0.4 ml aliquots werecounted in a 1272 CliniGamma counter (LKB). Cell counts determined by aCoulter Counter were employed to establish the number of cells per well.Background counts were subtracted from all samples. Scatchard analysisof the specifically bound material vs. the molar amount of coldcompetitor was carried out using Origin 5.0 software (Microcal Software,Inc., Northampton, Mass.). All experiments were carried in triplicateand repeated at least twice.

B. Empirical Data and Results Experimental Series I

This series of experiments is directed to demonstrating the role of cellassociated heparan sulfate chimeric core proteins in endothelial cellsin-situ. The bulk of the experiments and empirical data in this seriesare in-vitro results.

Experiment 1:

The immortalized human endothelial cell line ECV304 was transfected withprepared retroviral constructs containing full length cDNAs for eithersyndecan-4 or glypican-1. In addition, in order to differentiatepotential biological effects secondary to increased mass of cell surfaceand/or extracellular heparan sulfates versus increased core proteinexpression, two additional chimera core protein constructs were created.In one, S4-GPI, syndecan 4 extracellular domain was linked to theglypican 1 GPI anchored; and in another, G1-S4c, the extracellulardomain of glypican 1 was linked to the transmembrane and cytoplasmicdomains of syndecan-4. Cells transfected with a vector only construct(ECV-VC) were used as control. Increased expression of both syndecan-4and glypican-1 constructs was expected to result in larger numbers ofheparan sulfate chains on the cell surface.

Subsequently, the total mass of heparan sulfate chains on the wild typeas well as the 4 newly generated transfected ECV cell lines wasdetermined. Total heparan sulfate mass was significantly increased (perμg of total cellular protein) in ECV-S4, ECV-G1, ECV-S4-GPI andECV-G1-S4c but not ECV-VC cells. This data is presented by Table E1.

In order to assess whether these changes in HS expression resulted inselective alterations of heparan binding growth factors, the lowaffinity binding of bFGF, a prototypical heparin binding growth factorwas examined. Scatchard analysis of the wild type and newly generatedtransfected ECV cell lines showed that there were no significant changesin the affinity of bFGF binding (see Table E2; mean of 3 experiments).At the same time, there was a 2-fold increase in the number of bFGFbinding sites in S4 and C1-S4c clones and somewhat smaller increase inECV-G1 and ECV-S4-GPI clones (Table E2). The smaller increase incell-associated HS mass in glypican and syndecan-4 GPI overexpresserswas expected given higher shedding rates for GPI-linked glypicancompared to the transmembrane syndecan. Also, the increase in the numberof bFGF binding sites was of the same order as the increase in the totalHS cell mass—thus showing that there was no preferential creation ofbFGF binding sites and, there was no significant change in thebFGF-HS/HS ratio (calculated as ratio of a relative increase in thenumber of HS-bFGF sites per cell and a relative increase in the total HSmass). Thus, for a ECV-S4 clone compared to control, there was a5.94*106/2.32*106=2.56 fold increase in the number of bFGF-HS sites(Table E2) and a 0.33/0.14=2.36 increase in the total HS mass (per Dgprotein, Table E1) giving the HS-bFGF/HS ratio of 2.36/2.56=0.75.

TABLE E1 HS Mass In Various Stable Clones ³⁵S HS/g protein ECV-VC 0.14 ±0.026 ECV-S4 0.33 ± 0.042 ECV-G1 0.23 ± 0.015 ECV-S4-GPI 0.24 ± 0.080ECV-G1-S4c 0.34 ± 0.050 ³⁵S counts in HS expressed per g of totalprotein.

TABLE E2 Effect of S4, G1 and chimera constructs expression on lowaffinity Kd and the number of binding sites for bFGF Number of sitesHS-bFGF/Total Kd per cell HS Ratio ECV-VC 0.60 * 10⁻⁹ 2.32 * 10⁶ 1.00ECV-S4 0.85 * 10⁻⁹ 5.94 * 10⁶ 0.92 ECV-G1 0.81 * 10⁻⁹ 3.60 * 10⁶ 0.95ECV-S4-GPI 0.69 * 10⁻⁹ 3.80 * 10⁶ 0.96 ECV-G1-S4c 0.53 * 10⁻⁹ 4.89 * 10⁶0.87Experiment 2:

To study the effect of syndecan-4 and glypican-1 expression onendothelial cell growth, the ability of wild type and newly created ECVcell lines to grow in-vitro in response to serum and bFGF was analyzed.Experimentally, all cells were growth arrested for 48 hours and thenstimulated with 0.25% FBS supplemented with 25 ng/ml bFGF. The data isshown by FIG. 14 in which, V-V-vector control; G1: glypican-1 fulllength cDNA; S4-GPI; syndecan-4 extracellular domain linked to the GPIanchor; S4: full length syndecan-4 cDNA; G1—S4c: extracellular domain ofglypican-1 linked to syndecan-4 transmembrane/cytoplasmic domain.

As shown therein, the ECV-S4 and ECV-G1—S4c cells demonstrated a 4-foldincrease in cell number compared to ECV wild type or vector-transfected(MSCV) Cells. At the same time, growth of ECV-G1 or ECV-S4-GPI cells didnot differ significantly from wild type ECV cells. Even though bothECV-G1 and ECV-S4-GPI clones had somewhat smaller numbers of bFGF-HSbinding sites per cell, the absence of any significant change in bFGFgrowth response is out of proportion to the magnitude of HS-bFGFincrease.

Experiment 3:

To test the effect of these constructs expression on the cells abilityto form vascular structures, wild type and newly generated ECV cloneswere plated on Matrigel in 10% FBS-DMEM. Three days later, the presenceof definable structures (cords and rings) was assayed by lightmicroscopy. As in the case of in-vitro growth assays, ECV-S4 andECV-G1-S4c cells formed more numerous and denser vascular structurescompared to wild type ECV, ECV-G1 or ECV-S4-GPI cells. The results areshown by FIGS. 15A-15C.

As seen in FIGS. 15A-15C respectively, vector transduced ECV cells(MSCV) as well as ECV transduced with full length syndecan-4 and G1-S4cconstruct-carrying retroviruses were plated on growth factor depletedMatrigel supplemented with 25 ng/ml bFGF. Photographs of the gels weretaken 72 hours later. Note the presence of increased vascular networksand cell density in S4 and G1-S4c panels compared to MSCV panel.

Experiment 4:

To further analyze the effect syndecan, glypican, or syndecan/glypicanchimeras expression on biological behavior of endothelial cells, themigration of wild type and generated ECV cell lines migration towardsserum and bFGF in Boyden chamber assays was analyzed. Similar to thegrowth assay results, the cell lines expressing increased amounts ofsyndecan-4 or glypican-syndican-4 cytoplasmic tail chimeras demonstrateda significantly higher ability to migrate compared to wild type ECV orECV expressing glypican-1 or extracellular domain of syndecan-4 linkedto the glypican-1 GPI anchor. This is shown by FIG. 16.

Overall Conclusions:

The experiments demonstrate, therefore, that syndecan-4 expressionresulted in significant increase in bFGF-stimulated growth of EC in 2-Dand 3-D cultures as well as in enhanced migration towards the bFGFgradient. These results cannot be attributed to the increase in HS cellmass or preferential creation of low affinity (HS) bFGF binding sitesrather than increased syndecan-4 core protein expression, sinceoverexpression of glypican-1 while producing the same increase in HSmass did not produce increased growth and migration responses to bFGF.This conclusion is further supported by observation that whileglypican-S4 cytoplasmic domain chimera closely mimicked effects ofsyndecan-4 overexpression, syndecan-4-GPI chimera had no effect on bFGFresponses in these cells. Finally, while both syndecan-4 and glypican-1expression increased total HS cell mass there was no significant changein the number of low or high (data not shown) affinity HS bFGF bindingsites. Thus, increased expression of syndecan-4 cytoplasmic domain isassociated with increased responsiveness to bFGF stimulation as definedby cell growth and migration assays.

Experimental Series II

The second experimental series is directed to demonstrating the role ofclimeric cone proteins in stimulating angiogenesis under in-vivoconditions. The experiments and data presented hereinafter arerepresentative of clinical conditions and medical pathologies in livinghumans and animals.

Experiment 5:

To demonstrate the role and effect of chimeric cone protein inregulation of angiogenesis in-vivo, a rat myocardial infarction model[as reported in Li et al., Am. J. Physiol. 270: H1803-H1811 (1997)] wasadapted to in-vivo studies using mice.

In this model, ligation of a proximal coronary artery leads toreproducible infarction accompanied by peri-infarction angiogenesis thatcan be characterized in a number of ways including in-situhybridization, immunocytochemistry and morphometric analysis. Using thismodel, rapid (within 1 hour) induction of syndecan-4 gene expression inperi-infarct region that was dependent on the influx of blood-derivedmacrophages was demonstrated. A comparison of the extent of angiogenesisin macrophage-deficient homozygous op/op mice (low post-MI syndecanexpression) to that in the op/op mice treated with GM-CSF (thusrestoring macrophage population and syndecan-1/4 expression) revealed a4 fold increase in neovascularization in the latter as determined byBudR intake and morphometric analysis. This result is shown by FIGS.17A-17F respectively.

FIGS. 17A-17F show BudR uptake in op/op homozygous (−/−) andheterozygous (+/−) mice over 3 days time. Note the intense BudR uptakeby cells on the infarct periphery in (+/−) mice but not in (−/−) micewithin the per-infarct area on both day 1 and day 3 post-infarction.

Experiment 6:

To further link syndecan expression to enhanced angiogenic response inthese settings, transgenic mice lines were generated with cardiacmyocyte-specific expression of PR-39 peptide using A-MHC promoter. ThePR-39 peptide has been shown to increase both syndecan-1 and syndecan-4expression in-vitro in a variety of cell types. [See for example, Galloet al., Proc. Natl. Acad. Sci. USA 91: 11035-11039 (1994) and Li et al.,Circ. Res. 81: 785-796 (1997)].

Analysis of syndecan gene expression in PR-39 transgenic micedemonstrated marked increase in expression of syndecan-4 and glypican-1genes. This is shown by FIG. 18. Equally important, there was nodetectable expression of syndecan-1 in either wild type or transgenicmice (data not shown).

Immunocytochemical analysis with anti-CD31 antibody demonstratedincreased vascular density in PR-39 transgenics and the morphometricanalysis confirmed a 3 fold increase in the number of capillaries andsmall (<200 μm diameter) diameter vessels in these mice.

In particular, FIG. 18 shows a Northern blot analysis of gene expressionin PR-39 transgenic mice. The LV myocardium from the wild type (WT) andtwo PR-39 transgenic lines (A,B) mice was subjected to Northern blotanalysis. Note the increased syndecan-4 and glypican-1 expression inboth transgenic mice compared to WT mice.

Experiment 6:

To confirm the functional significance of this increase in vascularity,the total coronary resistance was assessed in an isolated heartpreparation as previously described [Li et al., J. Clin. Invest. 100:18-24 (1997)]. In these settings, a 2 fold decrease in coronaryperfusion pressure was observed for any given perfusion rate, thusconfirming a reduced transmyocardial resistance to flow. To furtherevaluate vascular function in these mice, a study of bFGF-inducedvasodilation in microvascular preparations in-vitro demonstrated anincreased bFGF sensitivity of PR-39 mice vessels. This is shown by thedata of FIG. 19.

As presented, FIG. 19 provides an in-vitro assessment of microvascularreactivity. Microvascular preparations from PR-39 transgenic and controlmice were preconstricted with endothelium and then evaluated for avasodilatory response to an endothelium-dependent agents ADP and bFGF.Note that while both PR-39 transgenics and controls are equallyresponsive to ADP, bFGF response is much more profound in the PR-39 mice(* p<0.05).

Overall Conclusions:

Myocardial-specific expression of PR-39 resulted in increased expressionof syndecan-4 and glypican-1 genes that was accompanied by afunctionally significant increase in coronary vascularity and enhancedbFGF responsiveness. These studies, therefore, provide rational evidenceand direct support for the in-vivo efficacy of climeric cone proteinexpression in angiogenic stimulation.

The present invention is not to be limited in scope nor restricted inform except by the claims appended hereto.

1. A prepared heterogeneous DNA segment for placement in an expressionvector capable of effecting transfection of endothelial cells in-situsuch that overexpression of extracellular matrix heparan sulphateproteoglycans subsequently occurs in-situ, said prepared heterogeneousDNA segment comprising: at least one first DNA sequence coding for theextracellular domain of a recombinant proteoglycan entity that is not anaturally occurring form of a syndecan-4 molecule, and which can beexpressed by a transfected endothelial cell in-situ after beingtransfected with an expression vector carrying said first DNA sequence,said extracellular domain first DNA sequence specifying theextracellular N-terminal portion of said expressed recombinantproteoglycan entity which then extends from the endothelial cell surfaceand is capable of binding heparan sulfates to form an extracellularmatrix in-situ; at least one second DNA sequence coding for thetransmembrane domain of said recombinant proteoglycan entity that is nota naturally occurring form of a syndecan-4 molecule, and which can beexpressed by a transfected endothelial cell in-situ after beingtransfected with an expression vector carrying said second DNA sequence,said transmembrane domain second DNA sequence specifying the medialportion of said expressed recombinant proteoglycan entity which thenextends through the endothelial cell membrane and is joined with saidextracellular N-terminal portion of said expressed recombinantproteoglycan entity; and at least one third DNA sequence coding for thecytoplasmic domain of a syndecan-4 molecule in said recombinantproteoglycan entity, and which can be expressed by a transfectedendothelial cell in-situ after being transfected with an expressionvector carrying said third DNA sequence, said syndecan-4 cytoplasmicdomain third DNA sequence specifying the cytoplasmic portion of saidexpressed recombinant proteoglycan entity which is then present withinthe cytoplasm of a transfected endothelial cell and is joined to saidtransmembrane portion of said expressed recombinant proteoglycan entity.2. The prepared heterogeneous DNA segment as recited by claim 1 whereinsaid first DNA sequence coding for the extracellular domain of saidrecombinant proteoglycan entity is selected from the group consisting ofsyndecan DNA sequences, glypican DNA sequences and perlecan DNAsequences.
 3. The prepared heterogeneous DNA segment as recited by claim1 wherein said second DNA sequence coding for the transmembrane domainof said recombinant proteoglycan entity is selected from the groupconsisting of syndecan DNA sequences, glypican DNA sequences andperlecan DNA sequences.
 4. A constructed expression vector capable ofeffecting transfection of endothelial cells in-situ such thatoverexpression of extracellular matrix heparan sulfate proteoglycansubsequently occurs in-situ, said constructed expression vectorcomprising: a prepared heterogeneous DNA segment comprised of (i) atleast one first DNA sequence coding for the extracellular domain arecombinant proteoglycan entity that is not a naturally occurring formof a syndecan-4 molecule, and which can be expressed by a transfectedendothelial cell in-situ after being transfected with an expressionvector carrying said first DNA sequence, said extracellular domain firstDNA sequence specifying the extracellular N-terminal portion of saidexpressed recombinant proteoglycan entity which then extends from theendothelial cell surface and is capable of binding heparan sulfates toform an extracellular matrix in-situ, (ii) at least one second DNAsequence coding for the transmembrane domain of said recombinantproteoglycan entity that is not a naturally occurring form of asyndecan-4 molecule, and which can be expressed by a transfectedendothelial cell in-situ after being transfected with an expressionvector carrying said second DNA sequence, said transmembrane domainsecond DNA sequence specifying the medial portion of said expressedrecombinant proteoglycan entity which then extends through theendothelial cell membrane and is joined with said extracellularN-terminal portion of said recombinant proteoglycan entity, and (iii) atleast one third DNA sequence coding for the cytoplasmic domain of asyndecan-4 molecule in said recombinant proteoglycan entity, and whichcan be expressed by a transfected endothelial cell in-situ after beingtransfected with an expression vector carrying said third DNA sequence,said syndecan-4 cytoplasmic domain third DNA sequence specifying thecytoplasmic portion of said expressed recombinant proteoglycan entitywhich is then present within the cytoplasm of a transfected endothelialcell and is joined to said transmembrane portion of said expressedrecombinant proteoglycan entity; and an expression vector carrying saidprepared heterogeneous DNA segment and capable of effecting transfectionof endothelial cells in-situ.
 5. The constructed expression vector asrecited by claim 4 wherein said expression vector capable of effectingtransfection of endothelial cells in-situ is a plasmid.
 6. Theconstructed expression vector as recited by claim 4 wherein saidexpression vector capable of effecting transfection of endothelial cellsin-situ is a virus.
 7. An in-situ transfected endothelial cell whichexists as a part of a living tissue in vitro, overexpressesextracellular matrix heparan sulfate recombinant proteoglycans, andpositions the recombinant proteoglycans at the endothelial cell surface,said in-situ transfected endothelial cell comprising: a viableendothelial cell transfected in-situ with a constructed expressionvector such that said transfected endothelial cell overexpressesextracellular matrix heparan sulfate recombinant proteoglycan entitiesthat are not a naturally occurring form of a syndecan-4 molecule, saidoverexpressed recombinant proteoglycan entities being comprised of (i)an extracellular N-terminal portion for said recombinant proteoglycanentity that is not a naturally occurring form of a syndecan-4 moleculeand which extends from the transfected endothelial cell surface andbinds heparan sulfates to form an extracellular matrix in-situ, saidextracellular N-terminal portion being the expressed product of at leastone first DNA sequence carried by the constructed expression vector andcoding for the extracellular domain of said recombinant proteoglycanentity expressed by the transfected endothelial cell in-situ, (ii) atransmembrane medial portion for said recombinant proteoglycan entitythat is not a naturally occurring form of a syndecan-4 molecule andwhich extends through the endothelial cell membrane and is joined withsaid extracellular N-terminal portion of said recombinant proteoglycanentity, said transmembrane medial portion being the expressed product ofat least one second DNA sequence carried by the constructed expressionvector and coding for the transmembrane domain of said recombinantproteoglycan entity expressed by the transfected endothelial cellin-situ, and (iii) a syndecan-4 cytoplasmic portion for said recombinantproteoglycan entity which is present within the cytoplasm of thetransfected endothelial cell and is joined to said transmembrane portionof said recombinant proteoglycan entity, said syndecan-4 cytoplasmicportion being the expressed product of at least one third DNA sequencecarried by the constructed expression vector and coding for thecytoplasmic domain of the syndecan-4 molecule of said recombinantproteoglycan entity expressed by the transfected endothelial cellin-situ.
 8. The in-situ transfected endothelial cell as recited by claim7 wherein said cell is selected from the group consisting of vascularendothelial cells and dermal endothelial cells.
 9. The in-situtransfected endothelial cell as recited by claim 7 wherein saidtransfected endothelial cell exists in a tissue comprising at least onekind of muscle cell selected from the group consisting of myocardialmuscle cells, smooth muscle cells and striated muscle cells.
 10. Amethod for making a prepared heterogeneous DNA segment intended forplacement in an expression vector capable of effecting transfection ofendothelial cells in-situ such that overexpression of extracellularmatrix heparan sulfate recombinant proteoglycan entities occurs in-situ,said method comprising the steps of: obtaining at least one first DNAsequence coding for the extracellular domain of a recombinantproteoglycan entity that is not a naturally occurring form of asyndecan-4 molecule, and which can be expressed by a transfectedendothelial cell in-situ after being transfected with an expressionvector carrying said first DNA sequence, said extracellular domain firstDNA sequence specifying the extracellular N-terminal portion of saidexpressed recombinant proteoglycan entity which then extends from thetransfected endothelial cell surface and is capable of binding heparansulfates to form an extracellular matrix in-situ; acquiring at least onesecond DNA sequence coding for the transmembrane domain of saidrecombinant proteoglycan entity that is not a naturally occurring formof a syndecan-4 molecule, and which can be expressed by a transfectedendothelial cell in-situ after being transfected with an expressionvector carrying said second DNA sequence, said transmembrane domainsecond DNA sequence specifying the medial portion of said expressedrecombinant proteoglycan entity which then extends through thetransfected endothelial cell membrane and is joined with saidextracellular N-terminal portion of said expressed recombinantproteoglycan entity; procuring at least one third DNA sequence codingfor the cytoplasmic domain of a syndecan-4 molecule in said recombinantproteoglycan entity, and which can be expressed by a transfectedendothelial cell in-situ after being transfected with an expressionvector carrying said third DNA sequence, said syndecan-4 cytoplasmicdomain third DNA sequence specifying the cytoplasmic portion of saidexpressed recombinant proteoglycan entity which is then present withinthe cytoplasm of an transfected endothelial cell and is joined to saidtransmembrane portion of said expressed recombinant proteoglycan entity;and joining together said extracellular domain first DNA sequence, saidtransmembrane domain second DNA sequence, and said syndecan-4cytoplasmic domain third DNA sequence as a prepared heterogeneous DNAsegment.
 11. A method for making a constructed expression vector capableof effecting transfection of endothelial cells in-situ such thatoverexpression of extracellular matrix heparan sulfate recombinantproteoglycan entities subsequently occurs in-situ, said methodcomprising the steps of: obtaining a prepared heterogeneous DNA segmentcomprised of (I) at least one first DNA sequence coding for theextracellular domain of a recombinant proteoglycan entity that is not anaturally occurring form of a syndecan-4 molecule, and which can beexpressed by a transfected endothelial cell in-situ after beingtransfected with an expression vector carrying said first DNA sequence,said extracellular domain first DNA sequence specifying theextracellular N-terminal portion of said expressed recombinantproteoglycan entity which then extends from the transfected endothelialcell surface and is capable of binding heparan sulfates to form anextracellular matrix in-situ, (ii) at least one second DNA sequencecoding for the transmembrane domain of said recombinant proteoglycanentity that is not a naturally occurring form of a syndecan-4 molecule,and which can be expressed by a transfected endothelial cell in-situafter being transfected with an expression vector carrying said secondDNA sequence, said transmembrane domain second DNA sequence specifyingthe medial portion of an expressed proteoglycan entity which thenextends through the transfected endothelial cell membrane and is joinedwith said extracellular N-terminal portion of said expressed recombinantproteoglycan entity, and (iii) at least one third DNA sequence codingfor the cytoplasmic domain of the syndecan-4 molecule in saidrecombinant proteoglycan entity, and which can be expressed by atransfected endothelial cell in-situ after being transfected with anexpression vector carrying said third DNA sequence, said syndecan-4cytoplasmic domain third DNA sequence specifying the cytoplasmic portionof an expressed proteoglycan entity which is then present within thecytoplasm of said transfected endothelial cell and is joined to saidtransmembrane portion of said expressed recombinant proteoglycan entity;and positioning said prepared heterogeneous DNA segment into anexpression vector suitable for transfection of endothelial cellsin-situ.